11第一讲_lecture_1_分子生物学的前世今生-学习资料

2024-07-28 作者:

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嗨，欢欢迎你跟我一起开开启分子生生物学之旅旅，人类从从分子水平平上认识生生命现象，，那也是经经历了遗传传学家，生生物化学家家和细胞生生物学家们们几代人人的共同努努力的，我我们从哪里里开始呢，嗯，让我我们先从遗遗传学之父父孟德尔先生说起起吧。 18655年，孟德德尔先生通通过对他在在庭院里种种植的豌豆的遗传传性状进行行观察，总结出了奠奠定遗传学学基础的性状遗传法性法则，他推推断生物体体的某一性性状是由一对因子子决定的。他的研究究结果在之之后的30多年中并并未被人们们所重视，，直到200世纪初，，的发现被另另外三位植植物学家分分别证实，人们才才认识到孟孟德尔研究究的价值。。孟德尔的这种遗传传因子在19091年被丹麦生物物学家约翰翰逊（Wilhhelm Ludwwig Johannnsen）赋赋予了“基基因”的名名称，除此此之外，遗遗传学中的基因型型和表型也也是约翰逊逊定义的。。但那个时时候人们还还不知道这这基因到底底是个什么么东东。 18800年细胞遗遗传学的奠奠基人德国生物学学家，华尔尔瑟∙弗莱明明（W. Fleemming）和爱德华华∙史特拉斯斯柏格(Edduard Strassburger)分别在动物分物和植物细细胞中发发现了有丝丝分裂(mitoosis)和后来来被我们称为染色色体的物质质，1902年，年Theodor Boveri和Walterr Sutton观观察到染色色体在减数数分裂过程程的分配规规律，并提提出了染色遗传因子的的色体是遗载体。 色体携带基基因的观点点就是遗传传学中的染色体理染论，这使得得基因不不再是非实实染色体的因子子，而有了了物质基础。这一一观点被当当时的一一些遗传学学家特别是摩尔根根Thomas HHunt Morrgan等人高度怀疑疑，有意思思的是，摩摩尔根本人人的研究却却在19100年为染色色体学说提提供了第一一个决定性性的证据。。摩尔根所所用的实实验材料是是果蝇，摩摩尔根发现现果蝇眼色色的表型式式性别连锁锁的。果蝇蝇具有个体体小，繁殖周期短短，后代数数 

第一生

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量多的优优点，实在在是比孟老老先生的豌豌豆更适宜宜的遗传学学研究模模式生物。但起初摩摩尔根实在在是不情愿愿得出这样样的结论，直到他对对残翅和黄黄体色两个个表型的的观察也发发现相同的的规律。这这才说服他他承认遗传传的染色体体理论。与此此同时，化化学家们也也没有闲着着，Friedrrich Miesscher早在在1869年就从白细年细胞中首次次分离的到到了一种他他称为“核核素”的化学物质，其实就是我其我们所说的DNA，1929年，菲巴斯•利文（Phhoebus Aaaron Levenne）分析出出了核素中含有四四种碱基和和分，并将其其命名为核酸核。不过过那时他认认为核酸是是由等量的各种碱碱基所组成成磷酸成分的重复四四联聚合物物，也就是是四核苷酸酸学说。19388年Rudolf Signer等人分析出等出DNA的分子量可的可以在5000‐1000kD之间，说明DNA是多酸的高分子子。但那时时利文的四四核苷酸学是普遍被人人是一种多聚核苷酸学说还是们所接受受的，没人人会想到这这种四个碱碱基重复排排列的DNNA可能会会是遗传物物质。 而其其实早在19281年Frrederick GGriffith就开开展了著名的细菌菌毒力转化化实验，他采用毒力力不同的肺肺炎双球菌菌注射给小小鼠，S型菌株能够型够使小鼠因感染死死亡，而无无毒的R型型肺炎双球球菌注射后后小鼠正常常存活，加热灭活的加的S型菌株株就失去去了使小鼠鼠致死的能能力，但当当把加热灭灭活的S型型菌株和正正常的R型菌株一一同注射给给小鼠时，，小鼠不幸幸升天，且且在其体内内分离得到到的居然是是S型的肺肺炎双球菌，这一一个实验开开始为“遗遗传物质是是DNA”提提供了实验验证据，但那时Griffith并没没有证明使R型菌菌株发生转转换的物质质是什么，直到19444年，Oswwald Avery，Colin MMacLeod和Maclynn McCarty三三位科学学家证明了了纯化了的的DNA可以使无毒的R型菌菌株发生转转化，而且且只有DNAA酶能够抑抑制这种转转化作用，蛋白酶酶或者RNAA都无法阻止转化化现象的发发 

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生。才逐逐渐让人们们开始接受受，DNA确确实是遗传传物质。1952年, A. D. Heershey 和Martha CChase通过过噬菌体侵侵染实验验再次用铁铁的证据给给人们展示示了DNA是遗传物物质。 了1950年Erwin Chaargaff通过过对不同生生物中各各种碱基含含量的分析析发现，不到了同生物中中的各种碱碱基的摩尔尔比是不一一样的，但但AT的摩摩尔比相同同，GC的摩尔比也的也总相同，这一发现现使得菲巴巴斯利文的的四核苷酸酸学说被推翻。并并为DNA双螺旋结结立奠定了基基础。 构的建立很快快，伦敦大学学国王学院的Rosaalind Elsiee Franklin和她的同和同事Mauricce Wilkinss采用X射射线衍射技技术得到了著名的的B型双螺螺旋衍射图图，“照片片51号””，而沃森森（James WWatson）和克里克克（Franciss Crick）在这张照片在片的启发下下很快构建了DNAA双螺旋模模型。至此此，让人们们倍感神秘秘的遗传物物质终于露露出了真面目。而而双螺旋模模型的建立立，也标志志着分子生生物学成为为了生命科科学领域的一门独独立的学科科。 自打打双螺旋结结构建立之之后，分子子生物学的的发展那叫叫一个日新月异，单从其互互不配对的的双链结构构克里克就就推测其和和遗传信息息的复制密切相关关，1957年又提出出了所谓中中心法则：DNA复制制产生DNA，DNA转录产生RNA，RNA翻译产生蛋翻蛋白质。基基本搭建了了狭义分子子生物学的的研究框架架。

先来来看看关于于复制。19958年, Maatthew MeselsonM和Franklinn Stahl用同位素标标记结合氯氯化铯密度度梯度离心心的办法，，证明了DNA的复复制是半保保留式的。1956年老Kornbeerg， Arthur Kornbberg就发现现了DNAA聚合酶。1968年冈崎令治治先生证明明了DNA复复制的半不连续方方式，19722年他又发发现DNAA新链合成成的起始需要RNAA 

第一生

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引物。 0年霍维兹兹等人发现现了RNA聚合酶，同样在二二十世纪660年代初初Marshall 1960Nirenbergg和Gobbind Khorana分别别解密了遗传密码遗，Francoiss Jacob和Sydneyy Brenner证证明了核糖糖体通过阅读mRNNA将遗传传信息翻译译成为蛋白白质。弗朗朗索瓦∙雅各布Franncois Jacoob和雅克∙莫诺Jacqques Monnod一同发发现了细菌菌基因转转录的调控控机制，也就就是著名的操纵子子模型。到到了70年代代，霍华德德∙马丁∙特特明Howaard Martinn Temin 戴戴维∙巴尔的的摩Davidd Baltimorre分别发现了反转转录酶，为克克里克提提出的中心心法则描实实了重要的的一笔。 70年年代后，分分子生物学学的发展可可以用井喷喷来形容，人们也从从初步认认识生命的的本质规律律进入到开开始改造生生命的阶段段。Paul Beerg在70年代建立年立了重组DNAD技术，堪称现代代遗传工程程之父。1975年时时，弗雷德德里克∙桑格格建立了一一种称为为链终止法法（chain tterminatioon methood）的技术术来测定DNA序列列，也称称做“双脱氧氧终止法”（Dideoxyy terminaation methhod）或是是“桑格法法”。两年之之后，他利利用此技技术成功定定序出Φ‐XX174噬菌体（Phagge Φ‐X1744）的基因因组序列。。这也是首首次完整整的基因组组定序工作作。桑格法法DNA测序技术成测成为人类基基因组计划划等研究得得以展开开的关键之之一，并使使桑格于19801年再再度获得诺诺贝尔化学学奖，桑格格真可谓是是生命科科学领域的的大牛，他他早在19555年就将将胰岛素的的氨基酸序序列完整地地定序出来来，同时时证明蛋白白质具有明明确构造。这项研究究使他单独独获得了1958年的的诺贝尔化化学奖。 19755年单克隆隆抗体技术建立，1975年德德国人G. J. F. Koohler、阿根根廷人C. Milstein和和丹麦科学学家N. K.. Jerne建立了单克克隆抗体技技术，他们们分享了19841年诺诺 

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贝尔生理理和医学奖奖。他们采采用产生抗抗体的单个个B淋巴细细胞同骨骨髓瘤细胞胞杂交，获获得既能产产生抗体，又能无限限增殖的杂杂种细胞，，并以此生生产针对抗抗原单一一表位的单单克隆抗体体，大大推推动了极微微量蛋白质质的检测技技术。这技技术也在生生物学和和医学的各各个领域中中得到广泛泛应用，并并为临床疾疾病的诊断断、治疗提供了新新手段。 19777年，Richhard J. Robberts和 PPhillip A. SharpS在研研究RNA转加工的过程程转录后加中，发现现了断裂基基因的存在在，人们也也由此开始始认识到，，RNA分子子在转录录后会将不不编码蛋白白质的所谓谓内含子部部分剪切掉掉。 19833年，凯利利·穆利斯斯博士（KKary Bankks Mullis ）发明了了PCR技术技，十年年后穆利斯斯因此获得得诺贝尔奖奖。PCR仪也成为为了现在几几乎每个生生命科学学实验室都都有的基本本设备。1993年获得得诺奖的另另一位前辈辈Michaeel Smith建建立了基基于核酸的的定点突变变技术，从从此人们得得以通过对对基因特定定位点的突突变来进行行基因功功能的研究究或对基因因进行定向向改造。

而到到了20世纪纪90年代代，人们已经经不再满满足于对基基因进行单单个了独立立的研究，，随着分子子生物学技技术的发展展，组学的的概念跃然然眼前。1990年起起，人类类基因组计计划拉开序序幕，并在在由公共基基金资助的的国际人类类基因组计计划和私人人企业美美国塞莱拉拉基因组公公司的激烈烈竞争中加加速完成， 2001年人类基基因组草图图发表，2003年，更为精细细的基因组组图谱公布布，2006年5月18日，英美美科学家宣宣布人类类最大和最最后一个染染色体———1号染色色体的基因因测序工作作已经完成成，历时时16年的的人类基因因组计划终终于画上了了句号。我我们也从此此完全的跨跨入了后基因组时时代。

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分子子生物学是是从遗传学学、细胞生生物学及生生物化学的基础上上发展起来来的，而他他自建立以以来，就一一刻不停的的在推动着着整个生命命科学的发发展，现在在很多的的生物学分分支学科上上都可以看看到时髦的的分子二字字。我们已已经超越了生命世世界的表层层现象，深深入到了分分子水平的的机制探索索，在关于于衰老、肿肿瘤、干细细胞、心血血管疾病病、记忆等等等生命医医学的各个个领域的研研究中，你你都会发现现分子生物物学无处处不在。同同时，我们们也正在应应用分子的的手段和技技术改变着着我们的生生命世界。从早期的克隆动动物，到如如今的合成成生物学，分子的技技术已经被被广泛的应应用到医疗疗、农业、环境和和社会生活活的各个方方面，那是是说也说不不过来的。且让我们们一点点开开始学习，感受分分子生物的的魅力和它它强大的力力量吧。

  附件：DNNA是遗传传物质的发发现过程

引自Esssential Ceell Biologyy (3th ediition) by AAlberts ett al. Garlaand Sciennce.

第一生

174HOW WE KNOW:GENES ARE MADE OF DNABy the 1920s, scientists generally agreed that genes

reside on chromosomes, and they knew that chromo-somes are composed of both DNA and proteins. But

because DNA is so chemically simple, researchers nat-urally assumed that genes had to be made of proteins,

which are much more chemically diverse. Even when

the experimental evidence suggested otherwise, this

assumption proved hard to es from the deadThe case for DNA began to take shape in the late 1920s,

when a British medical officer named Fred Griffith made

an astonishing discovery. He was studying Streptococcus

pneumoniae (pneumococcus), a bacterium that causes

pneumonia. As antibiotics had not yet been discovered,

infection with this organism was usually fatal. When

living S strain ofS. pneumoniaemouse injected

with S strain

mouse dies

living R strain ofS. pneumoniaemouse injected

with R strain

mouse lives

S strain

heat-killed

mouse injected mouse lives

living R strain+

mouse injected mouse dies living, pathogenic

S strain recoveredS strain

heat-killed

Figure 5–3 Griffith showed that heat-killed bacteria can transform living

cells. The bacterium Streptococcus

pneumoniae comes in two forms

that differ from one another in their

microscopic appearance and in their

ability to cause disease. Cells of the

pathogenic strain, which are lethal when

injected into mice, are encased in a

slimy, glistening polysaccharide capsule.

When grown on a plate of nutrients

in the laboratory, this disease-causing

bacterium forms colonies that look

dome-shaped and smooth; hence it is

designated the S form. The harmless

strain of the pneumococcus, on the other

hand, lacks this protective coat; it forms

colonies that appear flat and rough—hence, it is referred to as the R form. As

illustrated, Griffith found that a substance

present in the pathogenic S strain could

permanently change, or transform, the

nonlethal R strain into the deadly S are Made of DNA 175grown in the laboratory, pneumococci come in two

forms: a pathogenic form that causes a lethal infection

when injected into animals, and a harmless form that is

easily conquered by the animal’s immune system and

produces no the course of his investigations, Griffith injected vari-ous preparations of these bacteria into mice. He showed

that pathogenic pneumococci that had been killed by

heating were no longer able to cause infection. The

surprise came when Griffith injected both heat-killed

pathogenic bacteria and live harmless bacteria into the

same mouse. This combination proved lethal: not only

did the animal die of pneumonia, but Griffith found that

its blood was teeming with live bacteria of the patho-genic form (Figure 5–3). The heat-killed pneumococci

had somehow converted the harmless bacteria into the

lethal form. What’s more, Griffith found that the change

was permanent: he could grow these “transformed”

bacteria in culture, and they remained pathogenic. But

what was this mysterious material that turned harmless

bacteria into killers? And how was this change passed

on to progeny bacteria?S-strain cells

fractionation of cell-free

extract into classes of

molecules

RNA

protein DNA lipid carbohydrate

molecules tested for transformation of R-strain cells

R

strain

R

strain

S

strain

R

strain

R

strain

CONCLUSION: The molecule that

carries the heritable information

is DNA.

Blowing bubbles

Griffith’s remarkable finding set the stage for the experi-ments that would provide the first strong evidence

that genes are made of DNA. The American bacteri-ologist Oswald Avery, following up on Griffith’s work,

discovered that the harmless pneumococcus could be

transformed into a pathogenic strain in a culture tube

by exposing it to an extract prepared from the patho-genic strain. It would take another 15 years, however,

for Avery and his colleagues Colin MacLeod and Maclyn

McCarty to successfully purify the “transforming princi-ple” from this soluble extract and to demonstrate that

the active ingredient was DNA. Because the transform-ing principle caused a heritable change in the bacteria

that received it, DNA must be the very stuff of which

genes are 15-year delay was in part a reflection of the aca-demic climate—and the widespread supposition that

the genetic material was likely to be made of protein.

Because of the potential ramifications of their work, the

researchers wanted to be absolutely certain that the

transforming principle was DNA before they announced

their findings. As Avery noted in a letter to his brother,

also a bacteriologist, “It’s lots of fun to blow bubbles,

but it’s wiser to prick them yourself before someone

else tries to.” So the researchers subjected the trans-forming material to a battery of chemical tests (Figure

5–4). They found that it exhibited all the chemical prop-erties characteristic of DNA; furthermore, they showed

that enzymes that destroy proteins and RNA did not

Figure 5–4 Avery, MacLeod, and McCarty demonstrated that

DNA is the genetic material. The researchers prepared an

extract from the disease-causing S strain and showed that the

“transforming principle” that would permanently change the

harmless R-strain pneumococci into the pathogenic S strain is

DNA. This was the first evidence that DNA could serve as the

genetic its ability to transform bacteria, while enzymes

that destroy DNA inactivated it. And like Griffith before

them, the investigators found that their purified prep-aration changed the bacteria permanently: DNA from

the pathogenic species was taken up by the harmless

species, and this change was faithfully passed on to

subsequent generations of bacteria.

This landmark study offered rigorous proof that puri-fied DNA can act as genetic material. But the resulting

paper, published in 1944, drew remarkably little atten-tion. Despite the meticulous care with which these

experiments were performed, geneticists were not

immediately convinced that DNA is the hereditary

material. Many argued that the transformation might

have been caused by some trace protein contaminant

in the preparations. Or that the extract might contain a

mutagen that alters the genetic material of the harmless

bacteria—converting the bugs to the pathogenic form—rather than containing the genetic material itself.176 Genes Are Made of DNAChapter 5 DNA and ChromosomesVirus cocktailsThe debate was not settled definitively until 1952, when

Alfred Hershey and Martha Chase fired up their labora-tory blender and demonstrated, once and for all, that

genes are made of DNA. The researchers were study-ing T2—a virus that infects and eventually destroys the

bacterium E. coli. These bacteria-killing viruses behave

like little molecular syringes: they inject their genetic

material into the host cell, while the empty virus heads

remain outside the infected bacterium (Figure 5–5A).

Once inside the cell, the viral genes direct the formation

of new virus particles. In less than an hour, the infected

cells explode, spewing thousands of new viruses into

the medium. These then infect neighboring bacteria,

and the process begins again.

The beauty of T2 is that these viruses contain only two

kinds of molecules: DNA and protein. So the genetic

material had to be one or the other. But which? The

experiment was fairly straightforward. Because the

viral DNA enters the bacterial cell, while the rest of the

virus particle remains outside, the researchers decided

to radioactively label the protein in one batch of virus

and the DNA in another. Then, all they had to do was

follow the radioactivity to see whether viral DNA or

viral protein wound up inside the bacteria. To do this,

Hershey and Chase incubated their radiolabeled viruses

with E. coli; after allowing a few minutes for infection to

take place, they poured the mix into a Waring blender

and hit “puree.” The blender’s spinning blades sheared

the empty virus heads from the surfaces of the bacterial

cells. The researchers then centrifuged the sample to

separate the heavier, infected bacteria, which formed

a pellet at the bottom of the centrifuge tube, from

the empty viral coats, which remained in suspension

(Figure 5–5B).As you have probably guessed, Hershey and Chase

found that the radioactive DNA entered the bacterial

cells, while the radioactive proteins remained with the

empty virus heads. They found that the radioactive DNA

was also incorporated into the next generation of virus

particles.

This experiment demonstrated conclusively that viral

DNA enters bacterial host cells, whereas viral protein

does not. Thus, the genetic material in this virus had

to be made of DNA. Together with the studies done by

Avery, MacLeod, and McCarty, this evidence clinched

the case for DNA as the agent of heredity.(A) (B)

DNA labeled

virus

with

32P

genetic material:

protein or DNA?

CENTRIFUGE

E. coli

cell

protein labeled

with

35S

viruses allowed to

infect E. coli

viral headssheared offthe bacteriainfected bacteriacontain

32P butnot

35SFigure 5–5 Hershey and Chase showed definitively that genes are made of DNA. (A) The researchers worked with T2 viruses,

which are made entirely of protein and DNA. Each virus acts as a molecular syringe, injecting its genetic material into a bacterium;

the empty viral capsule remains attached to the outside of the cell. (B) To determine whether the genetic material of the virus is

protein or DNA, the researchers radioactively labeled the DNA in one batch of viruses with

32P and the proteins in a second batch

of viruses with

35S. Because DNA lacks sulfur and the proteins lack phosphorus, these radioactive isotopes provided a handy way

for the researchers to distinguish these two types of molecules. These labeled viruses were then allowed to infect E. coli, and the

mixture was disrupted by brief pulsing in a Waring blender to separate the infected bacteria from the empty viral heads. When the

researchers measured the radioactivity, they found that most of the

32P-labeled DNA had entered the bacterial cells, while the vast

majority of the

35S-labeled proteins remained in solution with the spent viral particles.